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. 2004 Feb 1;377(Pt 3):675–684. doi: 10.1042/BJ20030969

Detergents profoundly affect inhibitor potencies against both cyclo-oxygenase isoforms.

Marc Ouellet 1, Jean-Pierre Falgueyret 1, M David Percival 1
PMCID: PMC1223887  PMID: 14510637

Abstract

The sensitivity of Coxs (cyclo-oxygenases) to inhibition is known to be highly dependent on assay conditions. In the present study, the inhibitor sensitivities of purified Cox-1 and -2 were determined in a colorimetric assay using the reducing agent N, N, N ', N '-tetramethyl- p -phenylenediamine. With the detergent genapol X-100 (2 mM) present, the potencies of nimesulide, ibuprofen, flufenamic acid, niflumic acid and naproxen were increased over 100-fold against Cox-2 and titration curve shapes changed, so that maximal inhibition now approached 100%. Indomethacin, diclofenac and flosulide were not changed in potency. Similar effects of genapol were observed with inhibitors of Cox-1. DuP-697 and two analogues became more than 10-fold less potent against Cox-2 with genapol present. Tween-20, Triton X-100 and phosphatidylcholine, but not octylglucoside, gave qualitatively similar effects as genapol. Similar detergent-dependent changes in inhibitor potency were also observed using a [(14)C]arachidonic acid HPLC assay. The increases in potency of ibuprofen, flufenamic acid, isoxicam and niflumic acid towards Cox-2 and ibuprofen towards Cox-1 were accompanied by a change from time-independent to time-dependent inhibition. The interactions of Cox inhibitors has been described in terms of multiple binding step mechanisms. The genapol-dependent increase in inhibitor potency for ketoprofen was associated with an increase in the rate constant for the conversion of the initial enzyme-inhibitor complex to a second, more tightly bound form. The loss of potency for some inhibitors is probably due to inhibitor partitioning into detergent micelles. The present study identifies detergents as another factor that must be considered when determining inhibitor potencies against both Cox isoforms.

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Selected References

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  1. Aronoff David M., Boutaud Olivier, Marnett Lawrence J., Oates John A. Inhibition of prostaglandin H2 synthases by salicylate is dependent on the oxidative state of the enzymes. J Pharmacol Exp Ther. 2003 Feb;304(2):589–595. doi: 10.1124/jpet.102.042853. [DOI] [PubMed] [Google Scholar]
  2. Bayly C. I., Black W. C., Léger S., Ouimet N., Ouellet M., Percival M. D. Structure-based design of COX-2 selectivity into flurbiprofen. Bioorg Med Chem Lett. 1999 Feb 8;9(3):307–312. doi: 10.1016/s0960-894x(98)00717-3. [DOI] [PubMed] [Google Scholar]
  3. Benoit E., Delatour P., Olivier L., Caldwell J. (-)-R-fenoprofen: formation of fenoprofenyl-coenzyme A by rat liver microsomes. Biochem Pharmacol. 1995 May 26;49(11):1717–1720. doi: 10.1016/0006-2952(94)00417-k. [DOI] [PubMed] [Google Scholar]
  4. Boutaud Olivier, Aronoff David M., Richardson Jacob H., Marnett Lawrence J., Oates John A. Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H(2) synthases. Proc Natl Acad Sci U S A. 2002 May 14;99(10):7130–7135. doi: 10.1073/pnas.102588199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buttgereit F., Burmester G. R., Simon L. S. Gastrointestinal toxic side effects of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2-specific inhibitors. Am J Med. 2001 Feb 19;110 (Suppl 3A):13S–19S. doi: 10.1016/s0002-9343(00)00728-2. [DOI] [PubMed] [Google Scholar]
  6. Callan O. H., So O. Y., Swinney D. C. The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen. J Biol Chem. 1996 Feb 16;271(7):3548–3554. doi: 10.1074/jbc.271.7.3548. [DOI] [PubMed] [Google Scholar]
  7. Cann J. R., Hinman N. D. Hummel-Dreyer gel chromatographic procedure as applied to ligand-mediated association. Biochemistry. 1976 Oct 19;15(21):4614–4622. doi: 10.1021/bi00666a011. [DOI] [PubMed] [Google Scholar]
  8. Cannon G. W., Breedveld F. C. Efficacy of cyclooxygenase-2-specific inhibitors. Am J Med. 2001 Feb 19;110 (Suppl 3A):6S–12S. doi: 10.1016/s0002-9343(00)00681-1. [DOI] [PubMed] [Google Scholar]
  9. Chan C. C., Boyce S., Brideau C., Charleson S., Cromlish W., Ethier D., Evans J., Ford-Hutchinson A. W., Forrest M. J., Gauthier J. Y. Rofecoxib [Vioxx, MK-0966; 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone]: a potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles. J Pharmacol Exp Ther. 1999 Aug;290(2):551–560. [PubMed] [Google Scholar]
  10. Copeland R. A., Williams J. M., Giannaras J., Nurnberg S., Covington M., Pinto D., Pick S., Trzaskos J. M. Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11202–11206. doi: 10.1073/pnas.91.23.11202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Coughlan S. J., Davenport J. W., Hind G. Reactive blue 2 is a potent inhibitor of a thylakoid protein kinase. Eur J Biochem. 1991 Apr 23;197(2):467–471. doi: 10.1111/j.1432-1033.1991.tb15933.x. [DOI] [PubMed] [Google Scholar]
  12. Cromlish W. A., Payette P., Culp S. A., Ouellet M., Percival M. D., Kennedy B. P. High-level expression of active human cyclooxygenase-2 in insect cells. Arch Biochem Biophys. 1994 Oct;314(1):193–199. doi: 10.1006/abbi.1994.1429. [DOI] [PubMed] [Google Scholar]
  13. Egan R. W., Humes J. L., Kuehl F. A., Jr Differential effects of prostaglandin synthetase stimulators on inhibition of cyclooxygenase. Biochemistry. 1978 May 30;17(11):2230–2234. doi: 10.1021/bi00604a033. [DOI] [PubMed] [Google Scholar]
  14. Forghani F., Ouellet M., Keen S., Percival M. D., Tagari P. Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminol luminescence. Anal Biochem. 1998 Nov 15;264(2):216–221. doi: 10.1006/abio.1998.2842. [DOI] [PubMed] [Google Scholar]
  15. Gierse J. K., Koboldt C. M., Walker M. C., Seibert K., Isakson P. C. Kinetic basis for selective inhibition of cyclo-oxygenases. Biochem J. 1999 May 1;339(Pt 3):607–614. [PMC free article] [PubMed] [Google Scholar]
  16. Hanel A. M., Lands W. E. Modification of anti-inflammatory drug effectiveness by ambient lipid peroxides. Biochem Pharmacol. 1982 Oct 15;31(20):3307–3311. doi: 10.1016/0006-2952(82)90565-2. [DOI] [PubMed] [Google Scholar]
  17. Johnson A. R., Marletta M. A., Dyer R. D. Slow-binding inhibition of human prostaglandin endoperoxide synthase-2 with darbufelone, an isoform-selective antiinflammatory di-tert-butyl phenol. Biochemistry. 2001 Jun 26;40(25):7736–7745. doi: 10.1021/bi002343f. [DOI] [PubMed] [Google Scholar]
  18. Kulmacz R. J., Lands W. E. Stoichiometry and kinetics of the interaction of prostaglandin H synthase with anti-inflammatory agents. J Biol Chem. 1985 Oct 15;260(23):12572–12578. [PubMed] [Google Scholar]
  19. Kulmacz R. J. Prostaglandin G2 levels during reaction of prostaglandin H synthase with arachidonic acid. Prostaglandins. 1987 Aug;34(2):225–240. doi: 10.1016/0090-6980(87)90246-2. [DOI] [PubMed] [Google Scholar]
  20. Kurumbail R. G., Kiefer J. R., Marnett L. J. Cyclooxygenase enzymes: catalysis and inhibition. Curr Opin Struct Biol. 2001 Dec;11(6):752–760. doi: 10.1016/s0959-440x(01)00277-9. [DOI] [PubMed] [Google Scholar]
  21. Lanzo C. A., Sutin J., Rowlinson S., Talley J., Marnett L. J. Fluorescence quenching analysis of the association and dissociation of a diarylheterocycle to cyclooxygenase-1 and cyclooxygenase-2: dynamic basis of cyclooxygenase-2 selectivity. Biochemistry. 2000 May 23;39(20):6228–6234. doi: 10.1021/bi992761o. [DOI] [PubMed] [Google Scholar]
  22. Luong C., Miller A., Barnett J., Chow J., Ramesha C., Browner M. F. Flexibility of the NSAID binding site in the structure of human cyclooxygenase-2. Nat Struct Biol. 1996 Nov;3(11):927–933. doi: 10.1038/nsb1196-927. [DOI] [PubMed] [Google Scholar]
  23. Menguy T., Chenevois S., Guillain F., le Maire M., Falson P., Champeil P. Ligand binding to macromolecules or micelles: use of centrifugal ultrafiltration to measure low-affinity binding. Anal Biochem. 1998 Nov 15;264(2):141–148. doi: 10.1006/abio.1998.2854. [DOI] [PubMed] [Google Scholar]
  24. Mevkh A. T., Sud'ina G. F., Golub N. B., Varfolomeev S. D. Purification of prostaglandin H synthetase and a fluorometric assay for its activity. Anal Biochem. 1985 Oct;150(1):91–96. doi: 10.1016/0003-2697(85)90444-0. [DOI] [PubMed] [Google Scholar]
  25. Miki T., Orii Y. The reaction of horseradish peroxidase with hydroperoxides derived from Triton X-100. Anal Biochem. 1985 Apr;146(1):28–34. doi: 10.1016/0003-2697(85)90390-2. [DOI] [PubMed] [Google Scholar]
  26. Morgenstern R. A simple alternate substrate test can help determine the aqueous or bilayer location of binding sites for hydrophobic ligands/substrates on membrane proteins. Chem Res Toxicol. 1998 Jun;11(6):703–707. doi: 10.1021/tx980013e. [DOI] [PubMed] [Google Scholar]
  27. Morrison J. F., Walsh C. T. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol. 1988;61:201–301. doi: 10.1002/9780470123072.ch5. [DOI] [PubMed] [Google Scholar]
  28. Ouellet M., Percival M. D. Effect of inhibitor time-dependency on selectivity towards cyclooxygenase isoforms. Biochem J. 1995 Feb 15;306(Pt 1):247–251. doi: 10.1042/bj3060247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ouellet M., Percival M. D. Mechanism of acetaminophen inhibition of cyclooxygenase isoforms. Arch Biochem Biophys. 2001 Mar 15;387(2):273–280. doi: 10.1006/abbi.2000.2232. [DOI] [PubMed] [Google Scholar]
  30. Percival M. D., Ouellet M., Vincent C. J., Yergey J. A., Kennedy B. P., O'Neill G. P. Purification and characterization of recombinant human cyclooxygenase-2. Arch Biochem Biophys. 1994 Nov 15;315(1):111–118. doi: 10.1006/abbi.1994.1478. [DOI] [PubMed] [Google Scholar]
  31. Picot D., Loll P. J., Garavito R. M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature. 1994 Jan 20;367(6460):243–249. doi: 10.1038/367243a0. [DOI] [PubMed] [Google Scholar]
  32. Riendeau D., Percival M. D., Boyce S., Brideau C., Charleson S., Cromlish W., Ethier D., Evans J., Falgueyret J. P., Ford-Hutchinson A. W. Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor. Br J Pharmacol. 1997 May;121(1):105–117. doi: 10.1038/sj.bjp.0701076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Riendeau D., Percival M. D., Brideau C., Charleson S., Dubé D., Ethier D., Falgueyret J. P., Friesen R. W., Gordon R., Greig G. Etoricoxib (MK-0663): preclinical profile and comparison with other agents that selectively inhibit cyclooxygenase-2. J Pharmacol Exp Ther. 2001 Feb;296(2):558–566. [PubMed] [Google Scholar]
  34. Rome L. H., Lands W. E. Structural requirements for time-dependent inhibition of prostaglandin biosynthesis by anti-inflammatory drugs. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4863–4865. doi: 10.1073/pnas.72.12.4863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Selinsky B. S., Gupta K., Sharkey C. T., Loll P. J. Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations. Biochemistry. 2001 May 1;40(17):5172–5180. doi: 10.1021/bi010045s. [DOI] [PubMed] [Google Scholar]
  36. Smith C. J., Zhang Y., Koboldt C. M., Muhammad J., Zweifel B. S., Shaffer A., Talley J. J., Masferrer J. L., Seibert K., Isakson P. C. Pharmacological analysis of cyclooxygenase-1 in inflammation. Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13313–13318. doi: 10.1073/pnas.95.22.13313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith T., Leipprandt J., DeWitt D. Purification and characterization of the human recombinant histidine-tagged prostaglandin endoperoxide H synthases-1 and -2. Arch Biochem Biophys. 2000 Mar 1;375(1):195–200. doi: 10.1006/abbi.1999.1659. [DOI] [PubMed] [Google Scholar]
  38. Smith W. L., DeWitt D. L., Garavito R. M. Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem. 2000;69:145–182. doi: 10.1146/annurev.biochem.69.1.145. [DOI] [PubMed] [Google Scholar]
  39. Smith W. L., Langenbach R. Why there are two cyclooxygenase isozymes. J Clin Invest. 2001 Jun;107(12):1491–1495. doi: 10.1172/JCI13271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Smith William L., Song Inseok. The enzymology of prostaglandin endoperoxide H synthases-1 and -2. Prostaglandins Other Lipid Mediat. 2002 Aug;68-69:115–128. doi: 10.1016/s0090-6980(02)00025-4. [DOI] [PubMed] [Google Scholar]
  41. So O. Y., Scarafia L. E., Mak A. Y., Callan O. H., Swinney D. C. The dynamics of prostaglandin H synthases. Studies with prostaglandin h synthase 2 Y355F unmask mechanisms of time-dependent inhibition and allosteric activation. J Biol Chem. 1998 Mar 6;273(10):5801–5807. doi: 10.1074/jbc.273.10.5801. [DOI] [PubMed] [Google Scholar]
  42. Spinedi A., Pacini L., Limatola C., Luly P., Farias R. N. A study of human erythrocyte acetylcholinesterase inhibition by chlorpromazine. Biochem J. 1991 Sep 1;278(Pt 2):461–463. doi: 10.1042/bj2780461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Swinney D. C., Mak A. Y., Barnett J., Ramesha C. S. Differential allosteric regulation of prostaglandin H synthase 1 and 2 by arachidonic acid. J Biol Chem. 1997 May 9;272(19):12393–12398. doi: 10.1074/jbc.272.19.12393. [DOI] [PubMed] [Google Scholar]
  44. Timofeevski Sergei L., Prusakiewicz Jeffery J., Rouzer Carol A., Marnett Lawrence J. Isoform-selective interaction of cyclooxygenase-2 with indomethacin amides studied by real-time fluorescence, inhibition kinetics, and site-directed mutagenesis. Biochemistry. 2002 Jul 30;41(30):9654–9662. doi: 10.1021/bi0203637. [DOI] [PubMed] [Google Scholar]
  45. Walker M. C., Kurumbail R. G., Kiefer J. R., Moreland K. T., Koboldt C. M., Isakson P. C., Seibert K., Gierse J. K. A three-step kinetic mechanism for selective inhibition of cyclo-oxygenase-2 by diarylheterocyclic inhibitors. Biochem J. 2001 Aug 1;357(Pt 3):709–718. doi: 10.1042/0264-6021:3570709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wu Y., Zhou C., Roberts M. F. Stereocontrolled syntheses of water-soluble inhibitors of phosphatidylinositol-specific phospholipase C: inhibition enhanced by an interface. Biochemistry. 1997 Jan 14;36(2):356–363. doi: 10.1021/bi960602o. [DOI] [PubMed] [Google Scholar]
  47. van Ryn J., Pairet M. Clinical experience with cyclooxygenase-2 inhibitors. Inflamm Res. 1999 May;48(5):247–254. doi: 10.1007/s000110050455. [DOI] [PubMed] [Google Scholar]
  48. van der Donk Wilfred A., Tsai Ah-Lim, Kulmacz Richard J. The cyclooxygenase reaction mechanism. Biochemistry. 2002 Dec 31;41(52):15451–15458. doi: 10.1021/bi026938h. [DOI] [PubMed] [Google Scholar]

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